Plant pathogens are responsible for significant annual crop yield losses. One strategy for the control of plant pathogens is the use of resistant cultivars selected for, or developed by, plant breeders for this purpose. However, novel mechanisms for pathogen resistance can be implemented more quickly by molecular methods of crop protection than by traditional breeding methods. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
Plants rely heavily on a chemical and biological armory for their defense from a variety of pests and pathogens. Small cysteine-rich proteins that have been implicated in host defense and isolated from plant sources include defensins, thionins, and small antimicrobial proteins (AMP's). Cyclotides, also cysteine-rich molecules, have recently been recognized and characterized as being involved in host defense (Craik et al. (1999), J. Mol. Biol. 294: 1327-1336; Craik et al. (2000), Toxicon 39: 43-60). Cyclotide polypeptides are encoded by gene sequences, are produced as linear precursors, are cysteine-rich, and are capable of being cyclized via a peptide bond. Cyclotides display a diverse range of biological activities such as antibacterial activity, antifungal activity, anti-HIV activity, and uterotonic activity (Craik (2001), Toxicon 39: 1809-1813). Cyclotides have additionally been shown to possess insecticidal activity (Jennings et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98:10614-10619). Cyclized cyclotides differ from classical proteins in that they have no free N- or C-terminus due to their amide-circularized backbone.
Cyclotide polypeptides are derived from longer precursor proteins and thus both cleavage and cyclization steps are involved in the production of the cyclic backbone. The cyclic backbone of the cyclotide molecule typically ranges in size from 29 to 37 amino acid residues and has three disulfide bonds that form a cystine knot motif where two disulfide bonds and their connecting backbone strands form a ring that is threaded by the third disulfide bond. The mechanism(s) inherent to backbone cyclization is currently not known. One possibility is enzymatic or chemical involvement in both the backbone cleavage of the mature domain and the subsequent cyclization. The combined features of the cyclic cystine knot produce a unique protein fold that is topologically complex and has exceptional chemical and biological stability.
The majority of the plant cyclotides have been isolated from Rubiaceae and Violaceae plants (Gustafson et al. (1994), J. Nat. Prod. 116: 9337-9338; Gustafson et al. (2000), J. Nat. Prod. 63: 176-178; Witherup et al. (1994), J. Nat. Prod. 57: 1619-1625; Saether et al. (1995), Biochemistry 34, 4147-4158; Bokesch et al. (2001), J. Nat. Prod. 64: 249-250; Schopke et al. (1993), Sci. Pharm. 61: 145-153; Claeson et al. (1998), J. Nat. Prod. 61: 77-81; Göransson et al. (1999), J. Nat. Prod. 62: 283-286; Hallock et al. (2000), J. Org. Chem. 65: 124-128; Broussalis et al. (2001), Phytochemistry 58: 47-51). Recently, two members of a new sub-class of the cyclotide family have been discovered in Curcurbitaceae (Hernandez et al. (2000), Biochemistry 39: 5722-5730.; Felizmenio-Quimio et al. (2001), J. Biol. Chem. 276: 22875-22882; Heitz et al. (2001), Biochemistry 40: 7973-7983; Trabi and Craik, (2002), Trends in Biochem. Sci. 27: 132-138).
Cyclotides may be used in transgenic plants in order to produce plants with increased resistance to pathogens such as fungi, viruses, bacteria, nematodes, and insects. Thus, embodiments of the present invention solve needs for the enhancement of a plant's defensive response via a molecularly based mechanism which can be quickly incorporated into commercial crops.